The present invention relates generally to the field of cytology, and in particular to an improved growth medium and method for culturing epithelial cells. The invention also relates to cultured primary explant cells and cultures of epithelial cell lines.
A majority of human cancers arise in epithelial tissue. In the body, epithelial cells separate the internal living stromal tissue from the exterior environment. Epithelial cells exist either as stratified layers that directly face the exterior environment (such as the epidermis) or as a monolayer that can be folded into circular acini or ducts containing lumina.
An epithelial cell is functionally polarized with respect to the two environments it separates. The basal surface of the epithelial is connected to extracellular matrix material which is organized as a basal lamina adjacent to the extracellular matrix of the stromal. The apical surface is free of an apposed extracellular matrix layer, and serves as a boundary to molecular diffusion.
In mammary tissue, the polarity of normal epithelial cells apparently is directed by attachment of the cells to the basal surface. Tumor cells seem to lose this polarity, however. Metastatic epithelial tumor cells secrete hydrolytic enzymes that degrade the basal lamina before they invade the stroma of the breast. Those tumor cells that have degraded the basal lamina lose their polarity, while those cells that remain in contact with non-degraded basal lamina remain polarized.
This difference in morphology between normal and tumor epithelial cells in vivo cannot be reproduced in vitro for several reasons. First, it is very difficult to grow normal epithelial cells in culture at all. Second, because the normal cells grown in culture do not reproduce epithelial morphology observed in vivo, it is difficult to distinguish between normal and transformed cells in culture, although such distinctions are crucial to investigating early events in tumor development. For example, investigations of breast cancer have relied upon the difference in longevity between normal and transformed cells in culture; this difference is the senescence of normal cells and the immortal growth of tumor cells. These in vitro phenomena take time, however, and do, not have clear counterparts in vivo.
It would be most useful if epithelium tissue could be grown in vitro conveniently and would exhibit the morphological, physiological, and biochemical behavior characteristic of epithelial cells grown in vivo. These capabilities would allow investigation in vitro of both the development of epithelial cancer, in terms of, what triggers such a cancer and how it proceeds, and how better to treat or prevent it.
Because it is difficult to grow normal epithelial cells in culture, most cancer researchers utilize immortal cell lines which are derived from other sources. For example, most studies of breast cancer biology have relied upon a few cell lines which are primarily derived from human breast cancer metastases or rodent primary cells. The best known lines (for example, MCF-7 and MDA-MB231, see Table 1) were generated from metastasized, drug resistant tumors; the lines were established from samples taken from patients who had already undergone radiation therapy and chemotherapy. These tumor lines represent the extreme end of the tumor progression spectrum, and are therefore limited in their usefulness as research tools. In addition to the lack of early stage tumor lines, only one xe2x80x9cnormalxe2x80x9d breast cell line is available (MCF10, see Table 1), and it is not derived from epithelial cells.
Primary human normal and tumor-derived breast epithelial cells have only recently been cultured. While both types of cells are difficult to culture, breast carcinomas remain virtually intractable. For example, MDA-MB231 was derived from the 231st attempt to generate a cell line by placing human tumor cells into an immunodeficient mouse strain.
Those cell lines which are available suffer from several disadvantages. The lines represent highly invasive tumor cell types, since most are derived from pleural effusions. They do not represent different stages of tumor growth, as most represent late stage tumors, and they do not represent non-invasive tumor types such as Ductal Carcinoma In Situ (xe2x80x9cDCISxe2x80x9d). Furthermore, the lines have undergone many passages. Tumor cells are known for their genomic instability and may further diverge genetically even after establishment of a cell line. Thus, cell lines many generations removed from the original tumors may have undergone selective pressures and clonal evolution. This process can create cell lines that bear little genetic, biochemical, and morphological resemblance to the original tumor from which they are derived. Finally, very little information is available about the original sources of the previously available lines.
Human mammary epithelial cells are typically grown in monolayer culture on tissue culture plastic surfaces in medium containing low concentrations of either serum or pituitary extract. Attempts to grow these cells in a more physiological manner have involved coating the plastic culture surface with either rat tail collagen or a commercially available matrix, MATRIGEL(copyright) Matrix.
Conventional methodology for culturing human mammary epithelial involves three steps. The first is to isolate epithelial cells from the tissue sample, primarily by the use of digestive enzymes, resulting in free cells and cell clumps, which can be separated from non-epithelial material. The second step is to place the cells on a surface, which is typically plastic. The third step is to surround the cells with a medium, which typically contains minimal essential nutrients, such as sugars and amino acids, as well as serum (Band, V. and Sager, R (1989) Proc. Nat""l Acad Sci (USA) 86: 1249-1253) or pituitary extract (Hammond, S L, et al. (1984) Proc Nat""l Acad Sci (USA) 81: 5435-5439).
A recent improvement includes coating the culture surface with a material to mimic the extracellular matrix in vivo. The material currently used is rat tail collagen (Yang, J, et al. (1980) J Nat""l Cancer Inst 65: 337-343; Hall, H G, et al. (1982) Proc Nat""l Acad Sci USA 79: 4672-4676) or MATRIGEL(copyright) Matrix. Recent reports (Peterson, O W, et al. (1992) Proc Nat""l Acad Sci (USA) 89: 9064-9068; Bergstraesser, L M and Weitzman, S A (1993) Cancer Research 53: 2644-2654) indicate that normal human epithelium, grown on MATRIGEL(copyright) Matrix in a complex culture medium which included hormones and growth factors, formed three-dimensional luminal structures called xe2x80x9corganoids,xe2x80x9d while malignant cells remained as single cells and migrated through the MATRIGEL(copyright) Matrix. The xe2x80x9corganoidsxe2x80x9d appeared as large structures made of hundreds of cells and many cell layers thick. On the other hands this system did not form duct-like structures or other structures of increasing complexity. Also, the cultures were not long-lived, surviving for only seven days. These culture methods thus are of limited value for investigating normal epithelial tissue growth and the early events of epithelial tumor development. Such investigations appear to require the existence of viable epithelial stem cells which have the potential to differentiate in vitro into the complex architecture that is normally observed in vivo.
It is therefore an object of the present invention to provide an improved culture method which is simple and inexpensive, and which results in cultured epithelial tissue that is long-lived and that produces secondary structures that resemble epithelial structures in vivo. As an example, such structures in breast tissue are duct-like, branching and luminal. The culture method includes less stringent and elaborate initial tissue disaggregation techniques, an improved culture medium for use in the initial establishment of the cultures, the presence of extracellular matrix components in the culture chamber, and an optimal density of living cells in the culture chamber. The method can be used to produce primary explant cell cultures, secondary or extended explant cell cultures, or cultures of immortal cell lines from epithelial tissue. The cultures obtained by this method survive for at least 8 weeks, as a primary culture. Alternatively, the cultures may be frozen for subsequent use.
It is a further object of the present invention to provide an enhanced culture medium for use in culturing epithelial tissue. The culture medium contains fetal bovine serum, newborn calf serum, and rat serum. The sera are preferentially pretested to avoid that sera which results in spontaneous differentiation of cultured embryonic stem cells in the presence of LIF, an inhibitor of differentiation, as such sera presumably contain mycotoxins and other impurities. The medium may be further supplemented by providing non-essential amino acids and nucleosides. Further supplements may include growth factors and hormones, preferably estrogen and progesterone. The complexity of the medium induces epithelial attachment to a basal substrate and subsequent differentiation in vitro beyond what has been previously observed.
Yet another object of the present invention is to provide primary explant cell cultures, secondary or extended cell cultures, and cell lines derived from epithelial cells. The cultured primary explant cells produce secondary structures which resemble those in vivo, and are thus morphologically more similar to the organs from which they are derived. Thus, they are a better source of material for conducting studies of early tumorigenesis, for examining the effects of putative carcinogens on normal epithelial cells to determine their role in tumorigenesis, and for investigating the behavior of newly transformed epithelial cells leading to developing better treatments to target these cells. The cell lines are useful for evaluating the effectiveness of various cancer treatments.
The present invention provides an enhanced cell culture medium for culturing human epithelial cells comprising fetal bovine serum, newborn calf serum, and rat serum. The culture medium may be supplemented with non-essential amino acids and with nucleosides. The medium may be further supplemented with growth factors and with hormones.
The present invention further provides a method of culturing excised epithelial tissue, comprising the steps of providing physically disaggregated primary epithelial tissue, placing the disaggregated tissue in a culture chamber on a solid surface of basement membrane matrix components at a final concentration of about 2-50 mg/ml, and culturing the tissue in culture medium comprising fetal bovine serum, newborn calf serum, and rat serum. The initial medium may be removed and replaced with fresh medium.
The present invention further provides a method of producing a cell line derived from epithelial tissue source selected from the group consisting of breast, endometrium, and ovary, wherein the method is as described above, except that the initial culture medium which is removed is placed into a fresh culture chamber, such that the detached cells from the primary explant culture present in the culture medium establish a cell line in the chamber. Alternatively, the present invention provides a method of producing a cell line derived from epithelial tissue source selected from the group consisting of breast, endometrium, and ovary, wherein the method comprises the steps of providing cultured primary explant cells, treating the primary explant culture with trypsin such that free cells arise, and placing the free cells into a fresh culture chamber, such that the detached cells from the treated primary explant culture establish a cell line in the chamber. In both methods, the cultures intermediate to the primary explant culture and the established cell line are considered secondary, or extended, explant cultures.
The invention also provides cultured primary explant cells, cultured extended explant cells, and cultured cell lines that are derived from an epithelial tissue source selected from the group consisting from breast, endometrium or ovary, wherein said cells are produced by the methods described above.